An electrochemical immunosensor based on enzyme-encapsulated liposomes and biocatalytic metal deposition

A novel electrochemical immunosensor based on double signal amplification of enzyme-encapsulated liposomes and biocatalytic metal deposition was developed for the detection of human prostate specific antigen (PSA). Alkaline phosphatase (ALP)-encapsulated and detection antibody-functionalized liposomes were first prepared and used as the detection reagent. In the sandwich immunoassay, the model analyte PSA was first captured by anti-PSA capture antibody immobilized on the electrode and then sandwiched with the functionalized liposomes. The bound liposomes were then lysed with surfactant to release the encapsulated ALP, which served as secondary signal amplification means. ALP on the electrode surface initiated the hydrolysis of ascorbic acid 2-phosphate (AA-p) to produce ascorbic acid. The latter, in turn, reduced silver ions on the electrode surface, leading to deposition of the metal silver on the electrode surface. Linear sweep voltammetry (LSV) was chosen to detect the amount of the deposited silver. The results showed that the anodic stripping peak current was linearly dependent on the PSA concentration in the range of 0.01-100 ng mL⁻¹, and a detection limit as low as 0.007 ng mL⁻¹ can be obtained. Since the cut-off value of human PSA is 4 ng mL⁻¹, the proposed electrochemical immunosensor would be expected to gain widespread applications for the detection of PSA in clinical diagnosis.     

Highly-sensitive label-free electrochemical carcinoembryonic antigen immunosensor based on a novel Au nanoparticles–graphene–chitosan nanocomposite cryogel electrode

For the first time, a simple and highly sensitive label-free electrochemical carcinoembryonic antigen (CEA) immunosensor based on a cryogel electrode has been developed and tested. The as-prepared nanocomposite combined the advantages of the graphene, AuNPs and chitosan (AuNPs–GP–CS) together with the ease of preparing a cryogel coupled to a silver deposition, to act as a redox mediator, on a Au electrode. Under the optimal conditions, the decrease of the cyclic voltammetry (CV) silver peak current was proportional to the CEA concentration over a range of from 1.0 × 10⁻⁶ to 1.0 ng mL⁻¹ with a detection limit of 2.0 × 10⁻⁷ ng mL⁻¹. This AuNPs–GP–CS cryogel electrode gave a 1.7 times higher sensitivity and 25 times lower detection limit than the non-cryogel electrode. Moreover, the proposed electrochemical immunosensor exhibited good selectivity, reproducibility and stability. When applied to analyse clinical serum samples, the data determined by the developed immunosensor were in agreement with those obtained by the current hospital analysis system (enzyme linked fluorescent assay) (P > 0.05), to indicate that the immunosensor would be potentially useful for clinical diagnostics.     

Magnetic Fe3O4@Au composite-enhanced surface plasmon resonance for ultrasensitive detection of magnetic nanoparticle-enriched α-fetoprotein

Small molecules or analytes present at low concentrations are difficult to detect directly using conventional surface plasmon resonance (SPR) techniques because only small changes in the refractive index of the medium are typically induced by the binding of these analytes. Here, we present an amplification technique using core–shell Fe₃O₄@Au magnetic nanoparticles (MNPs) for an SPR bioassay. To evaluate this amplification effect, a novel SPR sensor based on a sandwich immunoassay was developed to detect α-fetoprotein (AFP) by immobilizing a primary AFP antibody (Ab₁) on the surface of a 3-mercapto-1-propanesulfonate/chitosan-ferrocene/Au NP (MPS/CS-Fc/Au NP) film employing Fe₃O₄@Au–AFP secondary antibody conjugates (Fe₃O₄@Au–Ab₂) as the amplification reagent. The stepwise fabrication of the biosensor was characterized using UV-vis spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. A calibration curve of Fe₃O₄@Au–Ab₂ conjugates amplification for AFP detection was obtained to yield a correlation in the range of 1.0–200.0 ng mL⁻¹ with a detection limit of 0.65 ng mL⁻¹, and a significant increase in sensitivity was therefore afforded through the use of Fe₃O₄@Au–Ab₂ conjugates as an amplifier. This magnetic separation and amplification strategy has great potential for the detection of other biomolecules of interest with low interference and high sensitivity by changing the antibody label used in the Fe₃O₄@Au–antibody conjugates.     

Ultrasensitive enzyme-free immunoassay for squamous cell carcinoma antigen using carbon supported Pd–Au as electrocatalytic labels

A novel nonenzymatic sandwich-type electrochemical immunosensor has been developed to detect squamous cell carcinoma antigen (SCCA). Nitrogen-doped graphene sheet (N-GS) was used to increase capacity of capturing primary antibodies (Ab₁). Carbon-supported Pd–Au binary nanoparticles (Pd–Au/C) were synthesized and used to label secondary antibodies (Ab₂). The specific binding of SCCA and antibodies enabled a quantitative attachment of Pd–Au/C on the electrode surface. Electrocatalytic analysis showed that the prepared Pd–Au/C exhibit excellent electrocatalytic activity towards hydrogen peroxide (H₂O₂). We use current response of electrocatalytic labels Pd–Au/C to detect the concentration of SCCA. The unique nonenzymatic immunosensor exhibits a relatively wide linear range from 0.005 to 2 ng mL⁻¹ and high sensitivity with a low detection limit of 1.7 pg mL⁻¹. The immunsensor also shows good reproducibility (4.2%) and stability (5.8%), which makes it an enormous application prospect in clinical research.     

An electrochemical biosensor for α-fetoprotein based on carbon paste electrode constructed of room temperature ionic liquid and gold nanoparticles

A novel and effective electrochemical immunosensor for the rapid determination of α-fetoprotein (AFP) based on carbon paste electrode (CPE) consisting of room temperature ionic liquid (RTIL) N-butylpyridinium hexafluorophosphate (BPPF₆) and graphite. The surface of the CPE was modified with gold nanoparticles for the immobilization of the α-fetoprotein antibody (anti-AFP). By sandwiching the antigen between anti-AFP on the CPE modified with gold nanoparticles and the secondary antibody, polyclonal anti-human-AFP labeled with horseradish peroxidase (HRP-labeled anti-AFP), the immunoassay was established. The concentration of AFP was determined based on differential pulse voltammetry (DPV) signal, which was generated in the reaction between O-aminophenol (OAP) and H₂O₂ catalyzed by HRP labeled on the sandwich immunosensor. AFP concentration could be measured in a linear range of 0.50-80.00 ng mL⁻¹ with a detection limit of 0.25 ng mL⁻¹. The immunosensor exhibited high sensitivity and good stability, and would be valuable for clinical assay of AFP.     

Immunosensor based on carbon nanotube/manganese dioxide electrochemical tags

This article reports on carbon nanotube/manganese dioxide (CNT–MnO₂) composites as electrochemical tags for non-enzymatic signal amplification in immunosensing. The synthesized CNT–MnO₂ composites showed good electrochemical activity, electrical conductivity and stability. The electrochemical signal of CNT–MnO₂ composites coated glassy carbon electrode (GCE) increased by nearly two orders of magnitude compared to bare GCE in hydrogen peroxide (H₂O₂) environment. CNT–MnO₂ composite was subsequently validated as electrochemical tags for sensitive detection of α-fetoprotein (AFP), a tumor marker for diagnosing hepatocellular carcinoma. The electrochemical immunosensor demonstrated a linear response on a log-scale for AFP concentrations ranging from 0.2 to 100 ng mL⁻¹. The limit of detection (LOD) was estimated to be 40 pg mL⁻¹ (S/N = 3) in PBS buffer. Further measurements using AFP spiked plasma samples revealed the applicability of fabricated CNT–MnO₂ composites for clinical and diagnostic applications.     

Ultrasensitive electrochemical immunosensor for zeranol detection based on signal amplification strategy of nanoporous gold films and nano-montmorillonite as labels

Nano-montmorillonites belong to aluminosilicate clay minerals with innocuity, high specific surface area, ion exchange, and favorable adsorption property. Due to the excellent properties, montmorillonites can be used as labels for the electrochemical immunosensors. In this study, nano-montmorillonites were converted to sodium montmorillonites (Na-Mont) and further utilized for the immobilization of thionine (TH), horseradish peroxidase (HRP) and the secondary anti-zeranol antibody (Ab₂). The modified particles, Na-Mont-TH-HRP-Ab₂ were used as labels for immunosensors to detect zeranol. This protocol was used to prepare the immunosensor with the primary antibody (Ab₁) immobilized onto the nanoporous gold films (NPG) modified glassy carbon electrode (GCE) surface. Within zeranol concentration range (0.01–12 ng mL⁻¹), a linear calibration plot (Y = 0.4326 + 8.713 X, r = 0.9996) was obtained with a detection limit of 3 pg mL⁻¹ under optimal conditions. The proposed immunosensor showed good reproducibility, selectivity, and stability. This new type of immunosensors with montmorillonites and NPG as labels may provide potential applications for the detection of zeranol.     

Nanosilver-doped DNA polyion complex membrane for electrochemical immunoassay of carcinoembryonic antigen using nanogold-labeled secondary antibodies

A simple and sensitive electrochemical immunoassay protocol was developed for the detection of carcinoembryonic antigen (CEA) using nanosilver-doped DNA polyion complex membrane (PIC) as sensing interface. To construct such an immunosensor, double-stranded DNA was initially assembled onto the surface of thionine/Nafion-modified screen-printed carbon electrode to adsorb silver ions with positive charges, then silver ions were reduced to nanosilver particles with the aid of NaBH₄, and then anti-CEA antibodies were immobilized on the nanosilver surface. Gold nanoparticles conjugated with horseradish peroxidase-labeled anti-CEA were employed as signal antibodies for the detection of CEA with a sandwich-type assay format. Under optimal conditions, the immunosensor exhibited a dynamic range of 0.03-32 ng mL⁻¹ with a low detection limit of 10 pg mL⁻¹ CEA. Intra- and inter-assay imprecision (CVs) were <9.5% and 6.5%, respectively. The response could remain 90.1% of the original current at 30th day. 50 real samples were evaluated using the immunosensor and the enzyme-linked immunosorbent assay, respectively, and received in accordance with those two methods.     

Signal amplification strategy for sensitive immunoassay of prostate specific antigen (PSA) based on ferrocene incorporated polystyrene spheres

A new kind of signal amplification strategy based on ferrocene (Fc) incorporated polystyrene spheres (PS-Fc) was proposed. The synthesized PS-Fc displayed narrow size distribution and good stability. PS-Fc was applied as label to develop immunosensors for prostate specific antigen (PSA) after the typical sandwich immunoreaction by linking anti-PSA antibody (Ab₂) onto PS-Fc. After the fabrication of the immunosensor, tetrahydrofuran (THF) was dropped to dissolve PS and release the contained Fc for the following stripping voltammetric detection. PS-Fc as a new electrochemical label prevented the leakage of Fc and greatly amplified the immunosensor signal. In addition, the good biocompatibility of PS could maintain the bioactivity of the antibodies. The response current was linear to the logarithm of PSA concentration in the range from 0.01 ng mL⁻¹ to 20 ng mL⁻¹ with a detection limit of 1 pg mL⁻¹. The immunosensor results were validated through the detection of PSA in serum samples with satisfactory results.     

Prussian blue–gold nanoparticles-ionic liquid functionalized reduced graphene oxide nanocomposite as label for ultrasensitive electrochemical immunoassay of alpha-fetoprotein

In this work, poly(diallyldimethylammonium chloride) (PDDA) protected Prussian blue/gold nanoparticles/ionic liquid functionalized reduced graphene oxide (IL-rGO-Au-PDDA-PB) nanocomposite was fabricated. The resulting nanocomposite exhibited high biocompatibility, conductivity and catalytic activity. To assess the performance of the nanocomposite, a sensitive sandwich-type immunosensor was constructed for detecting alpha-fetoprotein (AFP). Greatly enhanced sensitivity for this immunosensor was based on triple signal amplification strategies. Firstly, IL-rGO modified electrode was used as biosensor platform to capture a large amount of antibody due to its increased surface area, thus amplifying the detection response. Secondly, a large number of Au-PDDA-PB was conjugated on the surface of IL-rGO, which meant the enrichment of the signal and the more immobilization of label antibody. Finally, the catalytic reaction between H₂O₂ and the IL-rGO-Au-PDDA-PB nanocomposite further enhanced the signal response. The signals increased linearly with AFP concentrations in the range of 0.01–100 ng mL⁻¹. The detection limit for AFP was 4.6 pg mL⁻¹. The immunosensor showed high sensitivity, excellent selectivity and good stability. Moreover, the immunosensor was applied to the analysis of AFP in serum sample with satisfactory result.     

Ultrasensitive electrochemical immunoassay for carcinoembryonic antigen based on three-dimensional macroporous gold nanoparticles/graphene composite platform and multienzyme functionalized nanoporous silver label

Three-dimensional macroporous gold nanoparticles/graphene composites (3D-AuNPs/GN) were synthesized through a simple two-step process, and were used to modify working electrode sensing platform, based on which a facile electrochemical immunoassay for sensitive detection of carcinoembryonic antigen (CEA) in human serum was developed. In the proposed 3D-AuNPs/GN, AuNPs were distributed not just on the surface, but also on the inside of graphene. And this distribution property increased the area of sensing surface, resulting in capturing more primary antibodies as well as improving the electronic transmission rate. In the presence of CEA, a sandwich-type immune composite was formed on the sensing platform, and the horseradish peroxidase-labeled anti-CEA antibody (HRP-Ab₂)/thionine/nanoporous silver (HRP-Ab₂/TH/NPS) signal label was captured. Under optimal conditions, the electrochemical immunosensor exhibited excellent analytical performance: the detection range of CEA is from 0.001 to 10 ng mL⁻¹ with low detection limit of 0.35 pg mL⁻¹ and low limit of quantitation (LOQ) of 0.85 pg mL⁻¹. The electrochemical immunosensor showed good precision, acceptable stability and reproducibility, and could be used for the detection of CEA in real samples. The proposed method provides a promising platform of clinical immunoassay for other biomolecules     

Electrochemical immunosensor based on nanoporpus gold loading thionine for carcinoembryonic antigen

Nanoporous gold (NPG) has recently received considerable attention in analytical electrochemistry because of its good conductivity and large specific surface area. A facile layer-by-layer assembly technique fabricated NPG was used to construct an electrochemical immunosensor for carcinoembryonic antigen (CEA). NPG was fabricated on glassy carbon (GC) electrode by alternatively assembling gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) using 1,4-benzenedimethanethiol as a cross-linker, and then AgNPs were dissolved with HNO₃. The thionine was absorbed into the NPG and then gold nanostructure was electrodeposited on the surface through the electrochemical reduction of gold chloride tetrahydrate (HAuCl₄). The anti-CEA was directly adsorbed on gold nanostructure fixed on the GC electrode. The linear range of the immunosensor was from 10 pg mL⁻¹ to 100 ng mL⁻¹ with a detection limit of 3 pg mL⁻¹ (S/N = 3). The proposed immunosensor has high sensitivity, wide linear range, low detection limit, and good selectivity. The present method could be widely applied to construct other immunosensors.     

Quantum dots based electrochemiluminescent immunosensor by coupling enzymatic amplification for ultrasensitive detection of clenbuterol

An ultrasensitive electrochemiluminescence (ECL) immunosensor based on CdSe quantum dots (QDs) has been designed for the detection of clenbuterol. The immunosensor was fabricated by layer by layer and characterized with atomic force microscopic images (AFM) and electrochemical impedance spectra (EIS). In oxygen-saturated pH = 9.0 Tris-HCl buffer, a strong ECL emission of QDs could be observed during the cathodic process due to the H₂O₂ product from electrochemical reduction of dissolved oxygen. Upon the formation of immunocomplex, the second antibody labeled with horseradish peroxidase was simply immobilized on the electrode surface. The ECL emission decreased since steric hindrance of the immunocomplex slowed down the electron-transfer speed of dissolved oxygen, and also could be greatly amplified by an enzymatic cycle to consume the self-produced coreactant. Using clenbuterol as model analyte, the ECL intensity was determined by the concentration of competitive immunoassay of clenbuterol with a wide calibration in the range of 0.05 ng mL⁻¹ to 1000 ng mL⁻¹, and a low detection limit was 0.02 ng mL⁻¹. The immunosensor shows good stability and fabrication reproducibility. It was applied to detecting practical samples with the satisfactory results. This immunosensing strategy opens a new avenue for detection of residue and application of QDs in ECL biosensing.     

One step electrochemically deposited nanocomposite film of chitosan-carbon nanotubes-gold nanoparticles for carcinoembryonic antigen immunosensor application

A simple and controllable one-step electrodeposition method for the preparation of a chitosan-carbon nanotubes-gold nanoparticles (CS-CNTs-GNPs) nanocomposite film was used to fabricate an immunosensor for detection of carcinoembryonic antigen (CEA). The porous three-dimensional CS-CNTs-GNPs nanocomposite film, which offered a large specific surface area for immobilization of antibodies, exhibited improved conductivity, high stability and good biocompatibility. The morphology of the formed nanocomposite film was investigated by scanning electron microscopy (SEM), and the electrochemical behaviors of the immunosensor were characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Under the optimal conditions, the proposed immunosensor could detect CEA in two linear ranges from 0.1 to 2.0 ng mL⁻¹ and from 2.0 to 200.0 ng mL⁻¹, with a detection limit of 0.04 ng mL⁻¹. The immunosensor based on CS-CNTs-GNPs nanocomposite film as the antibody immobilization matrix could exhibit good sensitivity, stability, and reproducibility for the determination of CEA.     

An ultrasensitive streptavidin-functionalized carbon nanotubes platform for chemiluminescent immunoassay

An ultrasensitive chemiluminescent (CL) immunoassay system was developed for the detection of tumor marker. This sandwich CL assay method was for the first time designed based on a highly efficient streptavidin-functionalized multi-walled carbon nanotubes (MWCNTs) platform. The glass slide was firstly silylanized with 3-gycidoxypropyltrimethoxysilane (GPTMS) to generate surface epoxy group functionality. Subsequently, the MWCNTs/chitosan solution was mixed with streptavidin solution, and a certain amount of the resulting suspension was dropped on the surface of the epoxy-activated glass substrate to form a firm streptavidin-functionalized MWCNTs platform. The biofunctionalized-MWCNTs platform shows large reactive surface area and excellent biocompatibility. The capture antibody can be efficiently immobilized on the biosensing platform surface based on the highly selective recognition of streptavidin to biotinylated antibody. Using α-fetoprotein (AFP) as model analyte, the proposed method exhibits wide linear range of 0.001–0.1 ng mL⁻¹ with a low detection limit down to 0.52 pg mL⁻¹. The CL immunoassay system displays 7.9-fold increase in the detection sensitivity compared to the immunosensor without using MWCNTs. Moreover, the resulting immunosensor demonstrates excellent specificity, good reproducibility, and acceptable stability. This streptavidin-functionalized MWCNTs platform opens a novel and promising avenue for fabricating ultrasensitive CL immunoassay system.     

Ultrasensitive electrochemical immunosensors for multiplexed determination using mesoporous platinum nanoparticles as nonenzymatic labels

An ultrasensitive multiplexed immunoassay method was developed at a disposable immunosensor array using mesoporous platinum nanoparticles (M-Pt NPs) as nonenzymatic labels. M-Pt NPs were prepared by ultrasonic method and employed to label the secondary antibody (Ab₂) for signal amplification. The immunosensor array was constructed by covalently immobilizing capture antibody (Ab₁) on graphene modified screen printed carbon electrodes (SPECs). After the sandwich-type immunoreactions, the M-Pt-Ab₂ was bound to immunosensor surface to catalyze the electro-reduction of H₂O₂ reaction, which produced detectable signals for readout of analytes. Using breast cancer related panel of tumor markers (CA125, CA153 and CEA) as model analytes, this method showed wide linear ranges of over 4 orders of magnitude with the detection limits of 0.002 U mL⁻¹, 0.001 U mL⁻¹ and 7.0 pg mL⁻¹ for CA125, CA153 and CEA, respectively. The disposable immunosensor array possessed excellent clinical value in cancer screening as well as convenient point of care diagnostics.     

Development of an immunosensor for the detection of testosterone in bovine urine

In the presented work, a disposable immunosensor for the detection of testosterone, an endogenous steroid hormone, in bovine urine has been developed using screen-printed electrodes (SPEs). Due to concerns over the use of steroid hormones as growth promoters, the EU prohibits their use in food producing animals. Consequently, rigorous screening procedures have been implemented in all member states to detect the illegal administration of such compounds. Competitive immunoassays were developed, initially by enzyme linked immunosorbent assay (ELISA), and subsequently transferred to an electrochemical immunosensor format using disposable screen-printed carbon electrodes. Horseradish peroxidase (HRP) was the enzyme label of choice and chronoamperometric detection was carried out using a tetramethylbenzidine/hydrogen peroxide (TMB/H₂O₂) substrate system, at +100 mV. The EC₅₀ values obtained for the assay in buffer and urine gave relatively comparable results, 710 pg mL⁻¹ and 960 pg mL⁻¹, respectively. The linear range obtained for the assay in buffer extended from 0.03 ng mL⁻¹ to 40 ng mL⁻¹; while that in urine ranged from 0.03 ng mL⁻¹ to 1.6 ng mL⁻¹. The corresponding limits of detection (LOD) in buffer and urine were 26 pg mL⁻¹ and 1.8 pg mL⁻¹. Cross reactivity profiles of the antibody have been examined, with notable cross reactivities with 19-nortestosterone (11.6%) and boldenone (9.86%). Precision studies for the sensor demonstrated adequate reproducibility (CV < 13%, n = 3) and repeatability (CV < 9%, n = 3). Recovery data obtained showed good agreement between spiking studies and known concentrations of analyte. Sensors showed stability for 4 days at +4 °C. A sensitive, highly specific, inexpensive, disposable immunosensor, showing excellent overall performance for the detection of testosterone in bovine urine, has been developed.     

A streptavidin functionalized graphene oxide/Au nanoparticles composite for the construction of sensitive chemiluminescent immunosensor

In this work, a novel streptavidin functionalized graphene oxide/Au nanoparticles (streptavidin/GO/AuNPs) composite is prepared and for the first time used to construct sensitive chemiluminescent immunosensor for the detection of tumor marker. The streptavidin/GO/AuNPs composite and the immunosensor are characterized using scanning electron microscopy, static water contact angle measurement and electrochemical impedance spectroscopy. The biofunctionalized composite has large reactive surface area and excellent biocompatibility, thus the capture antibody can be efficiently immobilized on its surface based on the highly selective recognition of streptavidin to biotinylated antibody. Using α-fetoprotein (AFP) as a model, the proposed chemiluminescent immunosensor shows a wide linear range from 0.001 to 0.1 ng mL⁻¹ with an extremely low detection limit down to 0.61 pg mL⁻¹. The resulting AFP immunosensor shows high detection sensitivity, fast assay speed, acceptable detection and fabrication reproducibility, good specificity and stability. The assay results of serum samples with the proposed method are in an acceptable agreement with the reference values. This work provides a promising biofunctionalized nanostructure for sensitive biosensing applications.     

Amperometric carbohydrate antigen 19-9 immunosensor based on three dimensional ordered macroporous magnetic Au film coupling direct electrochemistry of horseradish peroxidase

A sandwich-type electrochemical immunosensor for the detection of carbohydrate antigen 19-9 (CA 19-9) antigen based on the immobilization of primary antibody (Ab₁) on three dimensional ordered macroporous magnetic (3DOMM) electrode, and the direct electrochemistry of horseradish peroxidase (HRP) that was used as both the label of secondary antibody (Ab₂) and the blocking reagent. The 3DOMM electrode was fabricated by introducing core–shell Au–SiO₂@Fe₃O₄ nanospheres onto the surface of three dimensional ordered macroporous (3DOM) Au electrode via the application of an external magnet. Au nanoparticles functionalized SBA-15 (Au@SBA-15) was conjugated to the HRP labeled secondary antibody (HRP-Ab₂) through the Au–SH or Au–NH₃⁺ interaction, and HRP was also used as the block reagent. The formation of antigen–antibody complex made the combination of Au@SBA-15 and 3DOMM exhibit remarkable synergistic effects for accelerating direct electron transfer (DET) between HRP and the electrode. Under the optimal conditions, the DET current signal increased proportionally to CA 19-9 concentration in the range of 0.05 to 15.65 U mL⁻¹ with a detection limit of 0.01 U mL⁻¹. Moreover, the immunosensor showed high selectivity, good stability, satisfactory reproducibility and regeneration. Importantly, the developed method was used to assay clinical serum specimens, achieving a good relation with those obtained from the commercialized electrochemiluminescent method.     

Construction of label-free electrochemical immunosensor on mesoporous carbon nanospheres for breast cancer susceptibility gene

In this contribution, mesoporous carbon nanospheres (MCN) were used to fabricate a label-free electrochemical immunosensor for breast cancer susceptibility gene (BRCAl). The detection platform was constructed by conjugation of anti-BRCA1 on glassy carbon electrodes which were modified by mesoporous carbon nanospheres–toluidine blue nanocomposite (MCN–TB)/room temperature ionic-liquid (RTIL) composited film. TB was adsorbed onto MCN and acted as a redox probe. The electroactivity of TB was greatly enhanced in the presence of MCN. The good conductivity of MCN and BMIM·BF₄ could promote the electron transfer and thus enhance the detection sensitivity. Moreover, the large surface area of MCN and the protein-binding properties of BMIM·BF₄ could greatly increase the antibody loading. The specific antibody–antigen immunoreaction on the electrode surface resulted in a decrease of amperometric signal of the electrode. Under optimized conditions, the amperometric signal decreased linearly with BRCAl concentration in the range of 0.01–15 ng mL⁻¹ with a low detection limit of 3.97 pg mL⁻¹. The immunosensor exhibits high sensitivity, good selectivity and stability.